Journal: eLife

Article Title: Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase

doi: 10.7554/eLife.82843

Figure Lengend Snippet: Early cholesterol synthesis overlaps with the mevalonate pathway, which produces farnesyl diphosphate and other isoprenoid precursors. Its rate-limiting enzyme is HMG-CoA reductase (HMGCR), the target of statins. The late cholesterol synthesis pathway is committed to sterol production and begins with squalene synthase (SQS). Squalene is converted to monooxidosqualene by the rate-limiting enzyme squalene monooxygenase (SM, grey) and cyclized by lanosterol synthase (LSS). Lanosterol is then converted by lanosterol 14α-demethylase (LDM) to testis meiosis-activating sterol (T-MAS), which is ultimately converted to cholesterol. A second round of SM-catalyzed epoxidation converts monooxidosqualene to dioxidosqualene, the precursor for a parallel ‘shunt’ pathway producing the regulatory oxysterol 24( S ),25-epoxycholesterol. Cholesterol synthesis is energy intensive and consumes a total of 11 O 2 molecules: one by SM, three by LDM, and seven by downstream enzymes. Double-headed arrows indicate multiple enzymatic steps.

Article Snippet: Chemical compound, drug , 2,3,22,23-Dioxidosqualene , Echelon Biosciences , S-0302 , .

Techniques:

Journal: eLife

Article Title: Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase

doi: 10.7554/eLife.82843

Figure Lengend Snippet: ( A ) HEK293T cells were incubated in medium containing fetal calf serum (FCS), lipoprotein-deficient FCS (LPDS) or LPDS containing 5 µM mevastatin and 50 µM mevalonolactone (LPDS +statin) for 8 hr, refreshed in their respective medium and incubated under normoxic or hypoxic conditions for 16 hr. ( B ) HEK293T cells were incubated under normoxic or hypoxic conditions for the indicated times. Non-saponifiable lipids were extracted, and squalene levels were determined using gas chromatography-mass spectrometry and adjusted relative to the normoxic condition, which was set to 1 (dotted line). The maximal squalene level detected was 0.66±0.12 ng per µg of total protein. ( C ) Pearson correlation between squalene levels in (B) and trunSM levels in . Blue line indicates linear regression. ( D ) HEK293T cells were treated with or without 300 µM squalene (squ.), monooxidosqualene (MOS) or dioxidosqualene (DOS) for 16 hr. ( E ) HEK293T SQLE -knockout ( SQLE -KO) clone 10 (c10) cells were transfected with the indicated constructs for 24 hr, then treated with or without 1 µM NB-598 or 300 µM squalene for 16 hr. ( A, D, E ) Graphs depict densitometric quantification of trunSM or truncated protein levels normalized to the (A) respective normoxic conditions for each serum type or (D, E) vehicle conditions, which were set to 1 (dotted line). ( A–E ) Data presented as mean ± SEM from n=3–5 independent experiments (*, p≤0.05; **, p≤0.01; [A] two-tailed ratio paired t -test; [D, E] two-tailed one-sample t -test vs. hypothetical mean of 1). Figure 4—source data 1. Uncropped immunoblots for .

Article Snippet: Chemical compound, drug , 2,3,22,23-Dioxidosqualene , Echelon Biosciences , S-0302 , .

Techniques: Incubation, Gas Chromatography, Mass Spectrometry, Knock-Out, Transfection, Construct, Two Tailed Test, Western Blot

Journal: eLife

Article Title: Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase

doi: 10.7554/eLife.82843

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , 2,3,22,23-Dioxidosqualene , Echelon Biosciences , S-0302 , .

Techniques: Generated, Stable Transfection, Expressing, Knock-Out, Transfection, Construct, Negative Control, Acid Assay, SYBR Green Assay, Western Blot, Software, Plasmid Preparation

Journal: bioRxiv

Article Title: Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase

doi: 10.1101/2022.08.18.504470

Figure Lengend Snippet: (A) HEK293T cells were incubated in medium containing fetal calf serum (FCS), lipoprotein-deficient FCS (LPDS) or LPDS containing 5 µM mevastatin and 50 µM mevalonolactone (LPDS + statin) for 8 h, refreshed in their respective media and incubated under normoxic or hypoxic conditions for 16 h. (B) HEK293T cells were incubated under normoxic or hypoxic conditions for the indicated times. Non-saponifiable lipids were extracted, and squalene levels were determined using gas chromatography-mass spectrometry and adjusted relative to the normoxic condition, which was set to 1 (dotted line). The maximal squalene level detected was 0.66 ± 0.12 ng per µg of total protein. (C) Pearson correlation between squalene levels in (B) and trunSM levels in . Blue line indicates linear regression. (D) HEK293T cells were treated with or without 300 µM squalene (Squ.), monooxidosqualene (MOS) or dioxidosqualene (DOS) for 16 h. (E) HEK293T SQLE -knockout ( SQLE -KO) clone 10 (c10) cells were transfected with the indicated constructs for 24 h, then treated with or without 1 µM NB-598 or 300 µM squalene for 16 h. (A, D, E) Graphs depict densitometric quantification of trunSM or truncated protein levels normalized to the (A) respective normoxic conditions or (D, E) respective vehicle conditions, which were set to 1 (dotted line). (A–E) Data presented as mean ± SEM from n ≥ 3 independent experiments (*, p ≤ 0.05; **, p ≤ 0.01; [A] two-tailed ratio paired t -test vs. FCS condition; [D, E] two-tailed one-sample t -test vs. hypothetical mean of 1).

Article Snippet: Chemicals were from the following suppliers: 2,3,22,23-dioxidosqualene (Echelon Biosciences S-0302), 2,3-oxidosqualene (monooxidosqualene; Echelon Biosciences S-0301), 5α-cholestane (Sigma-Aldrich C8003), bafilomycin A1 (Sigma-Aldrich B1793), BIBB 515 (Cayman Chemical 10010517), CB-5083 (Cayman Chemical 16276), DM-NOFD (Sigma-Aldrich SML1874), DMOG (Sigma-Aldrich D3695), FG-4592 (Cayman Chemical 15294), GR70585X (GlaxoSmithKlein), mevalonolactone (Sigma-Aldrich M4667), mevastatin (Sigma-Aldrich M2537), MG132 (Sigma-Aldrich C2211), NB-598 (Chemscene CS-1274), oleate-bovine serum albumin complexes (Sigma-Aldrich O3008), squalane (Sigma-Aldrich 234311), squalene (Sigma-Aldrich S3626), TAK-475 (Sigma-Aldrich SML2168).

Techniques: Incubation, Gas Chromatography, Mass Spectrometry, Knock-Out, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase

doi: 10.1101/2022.08.18.504470

Figure Lengend Snippet: (A) Simplified schematic of the cholesterol synthesis pathway depicting activities of squalene synthase (SQS), SM, lanosterol synthase (LSS) and lanosterol 14α-demethylase (LDM), alongside chemical structures of squalene, monooxidosqualene (MOS), dioxidosqualene (DOS), squalane, and farnesyl diphosphate. Double-headed arrows indicate multiple enzymatic steps, red labels indicate inhibitors of cholesterol synthesis, and green and blue labels indicate molecules that were determined to promote or not to promote trunSM accumulation, respectively. (B) Huh7 cells were treated with or without 300 µM squalene for 16 h. (C) HEK293T cells were treated with or without 300 µM squalane for 16 h. (D) Parental (WT) or HEK293T SQLE -knockout ( SQLE -KO) clone 10 (c10) cells were transfected with the indicated constructs for 24 h, then treated with or without 300 µM squalene for 16 h. (E) HEK SM-N100-GFP-V5 cells were treated with the indicated compounds under normoxic or hypoxic conditions for 16 h. (F) HEK MARCHF6-V5 cells were treated with or without 300 µM squalene or squalane for 16 h. (B–F) Graphs depict densitometric quantification of protein levels normalized to the respective vehicle or (E) respective normoxic conditions, which were set to 1 (dotted line). Data presented as mean ± SEM from n ≥ 3 independent experiments (*, p ≤ 0.05; two-tailed one-sample t -test vs. hypothetical mean of 1).

Article Snippet: Chemicals were from the following suppliers: 2,3,22,23-dioxidosqualene (Echelon Biosciences S-0302), 2,3-oxidosqualene (monooxidosqualene; Echelon Biosciences S-0301), 5α-cholestane (Sigma-Aldrich C8003), bafilomycin A1 (Sigma-Aldrich B1793), BIBB 515 (Cayman Chemical 10010517), CB-5083 (Cayman Chemical 16276), DM-NOFD (Sigma-Aldrich SML1874), DMOG (Sigma-Aldrich D3695), FG-4592 (Cayman Chemical 15294), GR70585X (GlaxoSmithKlein), mevalonolactone (Sigma-Aldrich M4667), mevastatin (Sigma-Aldrich M2537), MG132 (Sigma-Aldrich C2211), NB-598 (Chemscene CS-1274), oleate-bovine serum albumin complexes (Sigma-Aldrich O3008), squalane (Sigma-Aldrich 234311), squalene (Sigma-Aldrich S3626), TAK-475 (Sigma-Aldrich SML2168).

Techniques: Knock-Out, Transfection, Construct, Two Tailed Test

Journal:

Article Title: Human Pregnane X Receptor Activation and CYP3A4/CYP2B6 Induction by 2,3-Oxidosqualene:Lanosterol Cyclase Inhibition

doi: 10.1124/dmd.108.025130

Figure Lengend Snippet: Concentration-dependent abilities of squalene 2,3-oxide and squalene 2,3:22,23-dioxide treatments to activate PXR-responsive reporter expression in HepG2 cells and to bind to PXR in vitro. A, HepG2 cells were transiently transfected with pSG5-hPXR1, XREM-CYP3A4-Luc, and pRL-CMV and then incubated for 24 h with (top) medium alone (UT) or containing 0.1% DMSO (DM), 0.1% methanol (MeOH), 0.3 to 10 μM squalene 2,3-oxide (SO), or 0.3 to 10 μM squalene 2,3:22,23-dioxide (SDO) or (bottom) 10 μM Ro 48-8071 (Ro), 0.1 μM NB-598 (NB), Ro and DM, or Ro and NB alone (UT) or in combination with MeOH, SO, or SDO. After treatment, cells were harvested for measurement of firefly and Renilla luciferase activities. Normalized (firefly/Renilla) values are expressed as mean ± S.D. (n = 3 wells/treatment group). Groups not sharing a letter are significantly different from each other, p < 0.05. B, the LanthaScreen TR-FRET PXR (SXR) competitive binding assay was used to evaluate the abilities of SO and SDO (left) and Ro 48-8071 (right) to interact with PXR in vitro. T0901317 was included as a positive control PXR ligand (the same binding data for T0901317 are replicated in each panel). IC50 values and 95% confidence intervals (CIs) are shown. For the Ro 48-8071 data, the IC50 value is an estimate, and CI could not be calculated.

Article Snippet: Squalene 2,3-oxide (2,3-oxidosqualene, racemic) and squalene 2,3:22,23-dioxide (2,3,22,23-dioxidosqualene, mixture of diastereomers) were purchased from Echelon Biosciences (Salt Lake City, UT).

Techniques: Concentration Assay, Expressing, In Vitro, Transfection, Incubation, Luciferase, Competitive Binding Assay, Positive Control, Binding Assay